Reporter molecules are routinely used to monitor molecular events in the fields of biology, biochemistry, immunology, cell biology and molecular biology. For example, reporter molecules are employed in assays where the levels of the reporter molecule are due to transcription from a specific promoter linked to the reporter molecule. Reporter molecule assays can be used to study biological processes including gene expression, receptor activity, transcription factors, intracellular signaling, mRNA processing, and protein folding. Analysis of reporter molecules that are typically used in such assays includes detection of radioactive isotopes, enzyme activity, fluorescence, or luminescence. This technology is discussed generally by Akhavan-Tafti et al., in: “Bioluminescence and Chemiluminescence. Fundamentals and Applied Aspects. Proceedings of the 8th International Symposium on Bioluminescence and Chemiluminescence.” Cambridge, September 1994. Eds. Campbel, Kricka, Stanley. John Wiley and Sons 1994.
Luminescence in biological assays typically involves the activity of a luminogenic protein. Luminogenic proteins that are useful in assay systems include, but are not limited to, Renilla luciferase, Oplophorus luciferase, Vargula (Cypridina) luciferase, Gaussia luciferase, and aequorin. In a luminescent reaction, the interaction of a luminogenic protein with an appropriate molecule, referred to as a luminophore, produces light as one of the reaction products. The quantity of light (i.e., the number of photons) produced in the reaction, can be measured. This measurement may be used qualitatively to determine if a certain substance is or is not present in a sample. This measurement also may be used quantitatively to calculate the concentration of luminogenic protein and/or luminophore in the reaction.
Luminescent reactions can be used to detect very small quantities of a particular analyte, the substance being identified and measured in an analysis. For example, luminescent reactions can be used to detect and quantify proteases, lipases, phosphatases, peroxidases, glycosidases, various metabolites such as ATP or NADH, and reporter molecules. Luminescent reactions can also be used to detect and quantify analytes through binding interactions, such as those mediated by antibodies and nucleotide probes. Another application of luminescent reactions is bioluminescence resonance energy transfer (BRET), which can determine if two molecules are capable of binding each other or are co-localized in a cell (Angers et al., Proc. Natl. Acad. Sci. U.S.A. 97(7):3684-9, 2000). Typically, luminescent reactions can be used to detect an analyte present in a sample at less than about 1×10−16 molar, often less than 1×10−19 molar.
When using luminescence to measure an analyte, it is preferred that little or no light is produced by reactions that are not dependent on the presence of the analyte. For example, under assay conditions typically used for Renilla luciferase, light can generally be detected even when the luminogenic protein is not present. Luminescence that is not dependent on the catalytic activity of a luminogenic protein is termed autoluminescence. This autoluminescence is considered “background” since it does not provide meaningful information about the system but does contribute to the overall luminescent output. Autoluminescence can limit the usefulness of an analytical assay by reducing the ability to measure accurately the quantity of light resulting from the activity of the analyte (ie. “signal”). This can be especially problematic if the magnitude of the noise is significant relative to the magnitude of the actual signal. This measurement uncertainty can be quantified in terms of the ratio of the magnitudes of the signal (S) and the background (B), or signal-to-background ratio (S/B).
Autoluminescence can be caused, for example, by spontaneous oxidation of the luminophore. Also, addition to the assay system of various components, such as lipids (especially above the critical micelle concentration or CMC), hydrophobic proteins (especially those with a defined three-dimensional structure), and cells or other biological materials containing hydrophobic microenvironments, can greatly increase autoluminescence.
One class of luminophores that can exhibit autoluminescence is the coelenterazines. Coelenterazines interact with a variety of marine luciferases to produce light due to the oxidation of the coelenterazine to its respective coelenteramide. If this oxidation is facilitated by a luminogenic protein, then the photon corresponds to the interaction between the substrate and the protein, thus producing a signal. Coelenterazines can also contribute to the background due to spontaneous luminescent oxidation when in solution, even when there is no luminogenic protein present. In addition to producing background, this instability can also cause the luminescent signal to be short lived. This can result in a need to measure the luminescence of many samples in a short period of time, introducing further uncertainty into the overall analysis.
Modifications of coelenterazines have been investigated in attempts to modify their luminescent response. Properties of coelenterazines which have been affected include sensitivity to calcium ions (Ca2+) (Shimomura et al., Biochem. J. 296: 549-551, 1993; Shimomura et al., Biochem. J. 261: 913-920, 1989); sensitivity to superoxide anion (O2−) (Teranishi et al., Anal. Biochem. 249: 37-43, 1997); specificity for individual luminogenic proteins (Inouye et al., Biochem. Biophys. Res. Comm. 233: 349-53, 1997); and permeation of cell membranes (Shimomura, Biochem. J. 326: 297-298, 1997). Typically, the derivatives differ from natural coelenterazine through the identity of the substituents attached to the core imidazopyrazine structure. Despite their improvements in certain assay environments, these modified coelenterazines are not reported to avoid the problem of autoluminescence.
It is thus desirable to provide compositions which can function, either directly or indirectly, as luminophores and which provide for reduced autoluminescence under normal use conditions. These compositions may further exhibit increased stability relative to conventional luminophores. It is also desirable to provide compositions which are sensitive to substances other than luminogenic proteins. The interaction of such a composition with these substances could convert the composition into a luminophore. Such multi-functional compositions could thus provide a way to analyze non-luminogenic substances or processes through luminescent methods.